The APOBEC3 DNA cytosine deaminases are a fundamental part of the mammalian innate immune response. In particular, APOBEC3A mediates foreign DNA restriction in human cells with an outcome analogous to the protective effects of restriction enzymes in bacteria. This enzyme's expression levels are dramatically up-regulated by interferon, CpG oligos, transfected double-stranded DNA, and some DNA viruses. Foreign DNA recovered from APOBEC3A-expressing cells contains enormous levels of cytosine deamination with as many as two thirds of the cytosines converted to uracils (detected as C/G to T/A transition mutations). First, we will use model DNA substrates and biochemical approaches to test the hypothesis that APOBEC3A prefers double-stranded DNA substrates, as opposed to other family members that recognize single-stranded DNA. Second, subcellular localization and biochemical fractionation experiments will be used to test the hypothesis that chromosomal DNA is not targeted by APOBEC3A because catalytically active enzyme is localized exclusively to the cytoplasm or a cytoplasmic sub-compartment such as the endosomes. Finally, we propose to advance our preliminary studies on novel small molecules to identify those that specifically inhibit APOBEC3A activity in human cells. We anticipate that bona fide APOBEC3A inhibitors will make human cells more amendable to genetic engineering. Overall, we anticipate elucidating more of the APOBEC3A-mediated foreign DNA restriction mechanism and discovering lead compounds to transiently neutralize this innate defense and render cells more susceptible to genetic engineering.